Protein-deamidating enzyme, microorganism producing the same, gene encoding the same, production process therefor, and use thereof

ABSTRACT

A method for the production of an enzyme, which comprises culturing in a medium a strain that belongs to a bacterium classified into Cytophagales or Actinomycetes, or a new bacterium  Chryseobacterium  sp. No. 9670 belonging to the genus  Chryseobacterium , and has the ability to produce an enzyme having a property to deamidate amido groups in protein, thereby effecting production of the enzyme, and subsequently collecting the enzyme from the culture mixture and a method for the modification of protein making use of a novel enzyme which directly acts upon amido groups in protein, as well as a gene which encodes the enzyme, a recombinant vector which contains the gene, a transformant transformed with the vector and a method in which the transformant is cultured in a medium to effect production of the protein-deamidating enzyme and then the protein-deamidating enzyme is collected from the culture mixture.

This is a divisional application of application Ser. No. 10/815,744,filed Apr. 2, 2004, now U.S. Pat. No. 7,462,477 which is a divisionalapplication of application Ser. No. 09/727,769, filed Dec. 4, 2000, (nowU.S. Pat. No. 6,756,221), which is a Continuation-In-Part application ofU.S. application Ser. No. 09/324,910, filed Jun. 3, 1999 (now U.S. Pat.No. 6,251,651); the above noted prior application are all herebyincorporated by reference.

FIELD OF THE INVENTION

This invention relates to a novel enzyme, namely a novel enzyme whichacts upon side chain amido groups in protein and thereby releases sidechain carboxyl groups and ammonia, to a production process thereof andto a novel bacterium which produces the same. Particularly, it relatesto a method for the production of an enzyme having a property todeamidate amido groups in protein, which comprises culturing a bacterialstrain capable of producing an enzyme having a property to deamidateamido groups in protein, that belongs to Cytophagales or Actinomycetes,more particularly to the genus Chryseobacterium, Flavobacterium,Empedobacter, Sphingobacterium, Aureobacterium or Myroides, in a medium,thereby allowing the strain to produce the enzyme, and subsequentlycollecting the enzyme from the culture mixture. More particularly, itrelates to a method for the production of an enzyme having a property todeamidate amido groups in protein, which comprises culturing a newstrain Chryseobacterium sp. No. 9670 that belongs to the genusChryseobacterium, thereby allowing the strain to produce the enzyme, andsubsequently collecting the enzyme from the culture mixture. Theinvention also relates to a method for the modification of protein,which uses a novel enzyme having an activity to directly act upon amidogroups in protein. It also relates to an enzyme which has a property todeamidate amido groups in protein, to a gene which encodes the enzyme,to a vector which contains the gene, to a transformant transformed withthe vector, and to a method for the production of an enzyme having aproperty to deamidate amido groups in protein, which comprises culturingthe transformant in a medium, thereby allowing the transformant toproduce the enzyme, and subsequently collecting the enzyme from theculture.

BACKGROUND ART

Glutaminase and/or asparaginase are enzymes which hydrolyze glutamineand/or asparagine to convert them into glutamic acid and/or asparticacid and ammonia, and it is well known that these enzymes are obtainedfrom animals, plants and microorganisms. However, these enzymes areenzymes which act upon glutamine and/or asparagine in a specific fashionand cannot deamidate glutamine and/or asparagine in a peptide. Muchless, they cannot deamidate γ and/or β-amido groups of glutamine and/orasparagine in a protein having larger molecular weight than that of apeptide. Still less, they cannot act upon glutamine and/or asparaginebonded in a protein state.

Also, transglutaminase is known as an enzyme which acts upon amidogroups existing in a peptide state. This enzyme catalyzes the reactionof introducing an amine compound into protein by covalent bonding or thereaction of cross-linking the glutamine residue and lysine residue ofprotein via ε-(γ-glutamyl)lysine-peptide bonding, using the amido groupof peptide-bonded glutamine as an acyl donor and the amino group of theprimary amine as an acyl acceptor. It is known that, when amine orlysine does not exists in the reaction system or blocked, water acts asan acyl acceptor and the glutamine residue in peptide is deamidated tobecome glutamic acid residue, but since this enzyme is basically an acylgroup transferase as described above, cross-linking reaction occurs whenallowed to act on a usual protein and the reaction to deamidate proteindoes not occur, so that this enzyme is different from the enzyme of theinvention.

In addition, Peptide glutaminase I and peptide glutaminase II producedby Bacillus circulans are known as an enzyme which performs deamidationby acting upon glutamine bonded in peptide. It is known that the formeracts on the glutamine residue existing at the C terminal of peptide andthe latter acts on the glutamine residue existing in the peptide.However, these enzymes do not act upon a high molecular weight proteinand acts only upon a low molecular weight peptide [M. Kikuchi, H.Hayashida, E. Nakano and K. Sakaguchi, Biochemistry, vol. 10, 1222-1229(1971)].

Also, plural studies have been made to attempt to allow these enzymes(Peptide glutaminase I and II) to act upon a high molecular weightprotein rather than a low molecular weight peptide, and it has beenrevealed that these enzymes do not act on a high molecular weightprotein but act only on a protein hydrolysate peptide. Illustratively,Gill et al. have reported that each of Peptide glutaminase I and II doesnot act on milk casein and whey protein both in native form anddenatured form. They also have reported that, as a result of studies onactivities on protein hydrolysate, only Peptide glutaminase II actedonly on peptide having a molecular weight of 5,000 or less. (B. P. Gill,A. J. O'Shaughnessey, P. Henderson and D. R. Headon, Ir. J. Food Sci.Technol., vol. 9, 33-41 (1985)). Similar studies were carried out byHamada et al. using soy bean protein, and the result was consistent withthe result by Gill et al. That is, it was reported that these enzymesshowed deamidation percentage of 24.4 to 47.7% on soy bean peptide(Peptone), but did not substantially act on soy bean protein (0.4 to0.8%) (J. S. Hamada, F. F. Shih, A. W. Frank and W. E. Marshall, J. FoodScience, vol. 53, no. 2, 671-672 (1988)).

A series of these reports by Hamada et al. show data indicating thatpeptidoglutaminase derived from Bacillus circulans acts on proteinthough very slightly. On the other hand, Kikuchi et al. (M. Kikuchi, H.Hayashida, E. Nakano and K. Sakaguchi, Biochemistry, vol. 10, 1222-1229(1971) and Gill et al. (B. P. Gill, A. J. O'Shaughnessey, P. Hendersonand D. R. Headon, Ir. J. Food Sci. Technol., vol. 9, 33-41 (1985)) haveused the same enzyme derived from the same strain (Bacillus circulansATCC 21590) and reported that this enzyme acts on low molecular weightpeptide but does not act on protein. The present inventor has purifiedthe peptidoglutaminase derived from Bacillus circulans ATCC 21590 andconfirmed that the slight apparent deamidation activity on proteinreported by Hamada et al. is based on the action the enzyme upon peptideformed by the protease contaminated in the peptidoglutaminasepreparation.

There is a report suggesting the existence of an enzyme originating fromplant seed, which catalyzes deamidation of protein (I. A. Vaintraub, L.V. Kotova and R. Shara, FEBS Letters, vol. 302, 169-171 (1992)).Although this report observed ammonia release from protein using apartially purified enzyme sample, it is clear that this report does notprove the existence of the enzyme disclosed in the invention based onthe following reasons. That is, since a partially purified enzyme samplewas used, absence of protease activity was not confirmed, and no changein molecular weight of substrate protein after the reaction was notconfirmed, there remains a possibility that not one enzyme but pluralenzymes such as protease and peptidase acted on protein to releaseglutamine and/or asparagine as free amino acids and ammonia was releasedby glutaminase and/or asparaginase which deamidate these free aminoacids or a possibility that glutamine-containing low molecular weightpeptide produced in a similar way is deamidated by a peptideglutaminase-like enzyme. In addition, there is a possibility thatdeamidation occurred as a side-reaction by protease. In particular, itshould be noted that this report clearly describes that a glutaminaseactivity which acted on free glutamine to release ammonia was present inthe partially purified preparation used therein.

Accordingly, there is no report until now which confirmed the presenceof an enzyme which catalyzes deamidation of high molecular weightprotein, by purifying the enzyme as a single protein and isolating andexpressing the gene encoding the same.

In general, when carboxyl groups are formed by deamidation of glutamineand asparagine residues in protein, negative charge of the proteinincreases and, as the results, its isoelectric point decreases and itshydration ability increases. It also causes reduction of mutual reactionbetween protein molecules, namely, reduction of association ability, dueto increase in the electrostatic repulsion. Solubility and waterdispersibility of protein sharply increase by these changes. Also,increase in the negative charge of protein results in the change of thehigher-order structure of the protein caused by loosening of itsfolding, thus exposing the hydrophobic region buried in the proteinmolecule to the molecular surface. In consequence, a deamidated proteinhas amphipathic property and becomes an ideal surface active agent, sothat emulsification ability, emulsification stability, foamability andfoam stability of the protein are sharply improved.

Thus, deamidation of a protein results in the improvement of its variousfunctional characteristics, so that the use of the protein increasessharply (e.g., Molecular Approaches to Improving Food Quality andSafety, D. Chatnagar and T. E. Cleveland, eds., Van Nostrand Reinhold,New York, 1992, p. 37).

Because of this, a large number of methods for the deamidation ofprotein have been studied and proposed. An example of chemicaldeamidation of protein is a method in which protein is treated with amild acid or a mild alkali under high temperature condition. In general,amido groups of glutamine and asparagine residues in protein arehydrolyzed by an acid or a base. However, this reaction is nonspecificand accompanies cutting of peptide bond under a strong acid or alkalicondition. It also accompanies denaturation of protein to spoilfunctionality of the protein.

Because of this, various means have been devised with the aim oflimiting these undesired reactions, and a mild acid treatment (e.g., J.W. Finley, J. Food Sci., 40, 1283, 1975; C. W. Wu, S. Nakai and W. D.Powie, J. Agric. Food Chem., 24, 504, 1976) and a mild alkali treatment(e.g., A. Dilollo, I. Alli, C. Biloarders and N. Barthakur, J. Agric.Food Chem., 41, 24, 1993) have been proposed. In addition, the use ofsodium dodecyl sulfate as an acid (F. F. Shih and A. Kalmar, J. Agric.Food Chem., 35, 672, 1987) or cation exchange resin as a catalyst (F. F.Shih, J. Food Sci., 52, 1529, 1987) and a high temperature treatmentunder a low moisture condition (J. Zhang, T. C. Lee and C. T. Ho, J.Agric. Food Chem., 41, 1840, 1993) have also been attempted.

However, all of these methods have a difficulty in completelyrestricting cutting of peptide bond. The cutting of peptide bond is notdesirable, because it inhibits functional improvement of proteinexpected by its deamidation (particularly reduction of foam stability)and also causes generation of bitterness. Also, the alkali treatmentmethod is efficient in comparison with the acid treatment method, but ithas disadvantages in that it causes racemization of amino acids andformation of lysinoalanine which has a possibility of exerting toxicity.

On the other hand, some enzymatic deamidation methods have also beenattempted with the aim of resolving these problems of the chemicalmethods. Namely, a protease treatment method under a high pH (pH 10)condition (A. Kato, A. Tanaka, N. Matsudomi and K. Kobayashi, J. Agric.Food Chem., 35, 224, 1987), a transglutaminase method (M. Motoki, K.Seguro, A. Nio and K. Takinami, Agric. Biol. Chem., 50, 3025, 1986) anda peptide glutaminase method (J. S. Hamada and W. E. Marshall, J. FoodSci., 54, 598, 1989) have been proposed, but all of these three methodshave disadvantages.

Firstly, the protease method cannot avoid cutting of peptide bond as itsoriginal reaction. As described in the foregoing, cutting of peptidebond is not desirable.

In the case of the transglutaminase method, it is necessary tochemically protect ε-amino group of lysine residue in advance, in orderto prevent cross-linking reaction caused by the formation of isopeptidebond between glutamine and lysine, as the original reaction of theenzyme. When a deamidated protein is used in food, it is necessary todeamidate glutamine after protection of the ε-amino group with areversible protecting group such as citraconyl group, to remove theprotecting group thereafter and then to separate the deamidated proteinfrom the released citraconic acid. It is evident that these stepssharply increase the production cost and are far from the realization.

In the case of the peptidoglutaminase method, on the other hand, sincethis enzyme is an enzyme which originally catalyzes only deamidation oflow molecular weight peptide as described in the foregoing, its jointuse with protease is inevitable, so that it causes problems of formingbitter peptide and reducing functionality (particularly foam stability)similar to the case of the protease method.

In consequence, though the reaction selectivity due to high substratespecificity of enzymes is originally one of the greatest advantages ofthe enzymatic method, which surpasses chemical and physical methods, itis the present situation that the enzymatic method cannot be put intopractical use for the purpose of effecting deamidation of proteinbecause of the absence of an enzyme which does not generate sidereactions and is suited for the deamidation of high molecular weightprotein.

Under such circumstances, an enzyme capable of deamidating proteinwithout causing reduction of molecular weight of the protein has beencalled for in the field of food protein industry.

Accordingly, the inventor of the invention has established a screeningmethod which can be applied broadly to the microbial world and, by theuse of this method, succeeded in finding microorganisms which canproduce an enzyme capable of deamidating protein without causingreduction of molecular weight of the protein broadly in the microbialworld, and have accomplished the invention by carrying out culturing ofthe microorganisms and isolation and purification of the deamidatingenzymes.

SUMMARY OF THE INVENTION

In view of the above, the present inventor has conducted intensivestudies searching for an inexpensive microorganism as the source of anenzyme capable of directly acting upon amido groups which are bonded toprotein and thereby effecting deamidation of the protein, and, as aresult of the efforts, have established a screening method which can beapplied broadly to microorganisms. The inventor has found that a newbacterial strain belonging to the genus Chryseobacterium, newly isolatedfrom a soil sample, can produce an enzyme which exerts the deamidationfunction by directly acting upon amido groups in the bonded state inprotein without cutting peptide bond and cross-linking proteinmolecules. Thereafter, the inventor has completed the invention byfinding that protein deamidated by this enzyme obtained by the screeninghas excellent functionality. In this specification, an enzyme which hasthe aforementioned actions is called a “protein-deamidating enzyme”.

The inventor has then isolated and purified the protein-deamidatingenzyme, determined nucleotide sequence of a gene coding for theprotein-deamidating enzyme and confirmed that the protein-deamidatingenzyme can be produced using a transformant transformed with a vectorcontaining the gene.

Accordingly, the invention provides a method for the production of aprotein-deamidating enzyme, the protein-deamidating enzyme, a gene(i.e., polynucleotide) which encodes the protein deamidating enzyme, andthe like, in particular:

(1) An enzyme which has an activity to deamidate amido groups in aprotein.

(2) An enzyme which has an activity to deamidate amido groups in aprotein by directly acting upon the amido groups without cutting peptidebonds and without cross-linking a protein.

(3) The enzyme as described in (1) or (2) above, wherein said enzyme isderived from a microorganism.

(4) A polypeptide which comprises a polypeptide having an activity todeamidate amido groups in protein and having the amino acid sequence ofSEQUENCE NO. 6 shown in the Sequence Listing, wherein one or more ofamino acid residues of the amino acid sequence may be modified by atleast one of deletion, addition, insertion and substitution.

(5) A polypeptide which comprises a polypeptide having the amino acidsequence of SEQUENCE NO. 6 shown in the Sequence Listing.

(6) A nucleotide which encodes a polypeptide having an activity todeamidate amido groups in protein.

(7) A nucleotide which encodes a polypeptide having an activity todeamidate amido groups in protein by directly acting upon the amidogroups without cutting peptide bonds and without cross-linking aprotein.

(8) A nucleotide which comprises a nucleotide being selected from thefollowing nucleotides (a) to (g) and encoding a polypeptide having anactivity to deamidate amido groups in protein;

(a) a nucleotide which encode a polypeptide having the amino acidsequence of SEQUENCE NO. 6 shown in the Sequence Listing,

(b) a nucleotide which encodes a polypeptide having the amino acidsequence of SEQUENCE NO. 6 shown in the Sequence Listing, wherein one ormore amino acid residues of the amino acid sequence are modified by atleast one of deletion, addition, insertion and substitution,

(c) a nucleotide which has the nucleotide sequence of SEQUENCE NO. 5shown in the Sequence Listing,

(d) a nucleotide which has the nucleotide sequence of SEQUENCE NO. 5shown in the Sequence Listing, wherein one or more bases of thenucleotide sequence are modified by at least one of deletion, addition,insertion and substitution,

(e) a nucleotide which hybridizes with any one of the aforementionednucleotides (a) to (d) under a stringent condition,

(f) a nucleotide which has homology with any one of the aforementionednucleotides (a) to (d), and

(g) a nucleotide which is degenerate with respect to any one of theaforementioned nucleotides (a) to (f).

(9) A nucleotide which comprises a nucleotide encoding a polypeptidehaving the amino acid sequence of SEQUENCE NO. 6 shown in the SequenceListing.

(10) A recombinant vector which contains the nucleotide of any one of(6) to (9) above.

(11) A transformant transformed with the recombinant vector of (10)above.

(12) A method for producing an enzyme having an action to deamidateamido groups in protein, which comprises culturing the transformant of(11) above, thereby allowing said transformant to produce an enzymehaving an activity to deamidate amido groups in protein, andsubsequently collecting the enzyme having an activity to deamidate amidogroups in protein from the culture mixture.

(13) A recombinant polypeptide having an action to deamidate amidogroups in protein, which is obtained by the method of (11) above byculturing the transformant and collecting the polypeptide from saidculture mixture.

(14) A method for producing a novel enzyme, which comprises culturing amicroorganism in a nutrient medium, thereby allowing said microorganismto produce a novel enzyme having an activity to deamidate amido groupsin protein, and subsequently collecting said enzyme.

(15) A method for producing a novel enzyme having an activity todeamidate amido groups in protein, which comprises culturing amicroorganism in a nutrient medium, thereby allowing the microorganismto produce a novel enzyme which has an activity to deamidate amidogroups in protein by directly acting upon the groups without causingsevering of peptide bond and cross-linking of protein, and subsequentlycollecting said enzyme.

(16) The production process according to (14) or (15) above, wherein themicroorganism is a bacterium belonging to Cytophagales or Actinomycetes.

(17) The production process according to (14) or (15) above, wherein themicroorganism is a bacterium belonging to Flavobacteriaceae.

(18) The production process according to (14) or (15) above, wherein themicroorganism belonging to the genus selected from the group consistingof Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium,Aureobacterium and Myroides.

(19) The production process according to (14) or (15) above, wherein themicroorganism belonging to the genus Chryseobacterium.

(20) The production process according to (14) or (15) above, wherein themicroorganism is a strain Chryseobacterium sp. No. 9670 (FERM BP-7351).

(21) A method for modifying a protein or a peptide, which comprisesallowing an enzyme having an activity to deamidate amido groups inprotein or peptide by directly acting upon the groups without causingsevering of peptide bond and cross-linking of protein to react with aprotein or a peptide.

(22) A composition for use in modification of a protein or a peptide,which comprises an enzyme having an activity to deamidate amido groupsin protein or peptide by directly acting upon the groups without causingsevering of peptide bond and cross-linking of protein, as the activeingredient.

(23) An isolated microorganism Chryseobacterium sp. No. 9670 (FERMBP-7351).

(24) A method for improving functionality of a plant or animal proteinand/or peptide, which comprises allowing an enzyme having an activity todeamidate amido groups in protein and peptide by directly acting uponthe groups without causing severing of peptide bond and cross-linking ofprotein to react with the protein and/or peptide.

(25) A method for improving functionality of food containing a plant oranimal protein and/or peptide, which comprises allowing an enzyme havingan activity to deamidate amido groups in protein and peptide by directlyacting upon the groups without causing severing of peptide bond andcross-linking of protein to react with the food.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing results of SDS-polyacrylamide gelelectrophoresis of the purified protein-deamidating enzyme of Example 6.Lane 1 shows molecular weight marker proteins and lane 2 is the purifiedprotein-deamidating enzyme.

FIG. 2 is a graph showing time course of the changes in deamidationratio of proteins of Example 7. Closed circle indicates wheat gluten,open circle indicates caseinate, closed triangle indicates whey protein,open square indicates albumen protein and open triangle indicatessoybean protein.

FIG. 3 is a graph showing results of SDS-polyacrylamide gelelectrophoresis of the deamidated proteins of Example 7. Lanes 1, 4, 7and 10 are molecular weight marker proteins, lanes 2, 5, 8 and 11 arecontrol caseinate, whey protein, albumen protein and soybean protein inthat order and lanes 3, 6, 9 and 12 are deamidated caseinate, wheyprotein, albumen protein and soybean protein in that order.

FIG. 4 is a graph showing pH-solubility curve of the deamidated glutenof Example 10. Closed circle indicates deamidated wheat gluten and opencircle indicates control wheat gluten.

FIG. 5 is a graph showing pH-solubility curve of the deamidatedcaseinate of Example 10. Closed circle indicates deamidated caseinateand open circle indicates control caseinate.

FIG. 6 is a graph showing pH-solubility curve of the deamidated wheyprotein of Example 10. Closed circle indicates deamidated whey proteinand open circle indicates control whey protein.

FIG. 7 is a graph showing pH-solubility curve of the deamidated soybeanprotein of Example 10. Closed circle indicates deamidated soybeanprotein and open circle indicates control whey protein.

FIG. 8 is a graph showing pH-solubility curve of the deamidated albumenprotein of Example 10. Closed circle indicates deamidated albumenprotein and open circle indicates control albumen protein.

DETAILED DESCRIPTION OF THE INVENTION

The protein-deamidating enzyme of the invention is effective on theamido group of at least asparagine residue or glutamine residue inprotein, but its action site is not particularly limited, and it can beeffective on the amido group connected to other amino acid residues. Inthis connection, the term “protein” as used herein is not limited tosimple protein and it may also be protein complexes, e.g., withsaccharides or lipids. Also, the molecular weight of the protein is notparticularly limited and is generally 5,000 (50 residues) or more andpreferably in the range of from 10,000 to 2,000,000.

The protein-deamidating enzyme of the invention can also be used for thedeamidation of peptides having amido groups or derivatives thereof, inaddition to proteins. Examples of the peptides include those generallyhaving amino acid residues of from 2 to 50, and those which are used asnutrition-improving agents are preferable.

Thus, the protein-deamidating enzyme of the invention can use fromdipeptides to high molecular weight proteins, including polypeptides, asits substrates. In this connection, the term “polypeptides” as used inthis specification includes proteins.

A microorganism capable of producing the protein-deamidating enzyme ofthe invention can be screened, for example, in the following manner.That is,

1) an enrichment culturing is carried out by inoculating a microbialsource such as a soil sample into a medium which contains Cbz-Gln-Gly asthe sole nitrogen source,

2) next, the culture broth obtained in 1) is inoculated onto an agarmedium which contains Cbz-Gln-Gly as the sole nitrogen source to obtaina grown strain, and then

3) the thus obtained strain is cultured in an appropriate liquidnutrient medium to check the activity in the culture broth to releaseammonia from Cbz-Gln-Gly and casein.

Composition of the medium to be used in the enrichment culturing isoptionally selected in response to the microorganism to be cultured,except that it contains Cbz-Gln-Gly as the sole nitrogen source, andculturing temperature and other various conditions are also optionallyselected in response to the microorganism to be cultured. When themicroorganism to be cultured is a bacterium, the medium described inExample 1 of the invention, for example, can be used, and when themicroorganism to be cultured is a fungus or yeast, a medium in whichnitrogen sources are removed from Czapex-Dox liquid medium (3% sucrose,0.1% K₂HPO₄, 0.05% MgSO₄.7H₂O, 0.05% KCl, 0.0001% FeSO₄.7H₂O) or amodified SD medium (2% glucose, 0.17% Bacto Yeast Nitrogen base withoutamino acids and ammonium sulfate (Difco)) can be used in addition to themedium described in Example 1. Regarding the culturing for screeningalgae, it can be carried out in accordance, for example, with“Microalgae, biotechnology and microbiology” (Becker, E. W., pp. 9-46,1993, Cambridge University Press, Cambridge, United Kingdom). Culturingwith the nutrient medium in the above step 3) is carried out in the samemanner. Selection and practice of these conditions do not requireunnecessary, inadequate, broad and immoderate experiments for thoseskilled in the art.

When one of the strains obtained in this manner (strain No. 9670) wasidentified in accordance with Bergey's Manual of DeterminativeBacteriology, it was identified as a species belonging to the genusChryseobacterium. This strain was named Chryseobacterium sp. No. 9670and deposited on Nov. 29, 1999 with accession number FERM P-17664(transferred to FERM BP-7351 on Nov. 8, 2000) at National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Japan of which address is 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, 305-8566, Japan.

The strain No. 9670 is a Chryseobacterium sp., because it is Gramnegative, in rod shape, non-motile, aerobic, catalase positive andoxidase positive, and it forms an insoluble yellow to orange pigment.

REFERENCES

-   (1) Vandamme, P., J.-F. Bernardet, P. Segers, K. Kersters and B.    Holmes, 1994. New Perspective in the Classification of the    Flavobacteria: Description of Chryseobacterium gen. nov., Bergeyella    gen. nov., and Empedobacter nom. rev., Int. J. Syst. Bacteriol., 44:    827-831.-   (2) Holmes, B., R. J. Owen and T. A. McMeekin, 1984. Genus    Flavobacterium, Bergey, Harrison, Breed, Hammer and Huntoon, 1923,    97^(AL), pp. 353-361. In N. R. Krieg and J. G. Holt (ed.), Bergey's    manual of systematic bacteriology, vol. 1, The Williams & Wilkins    Co., Baltimore.    I. Morphology

Shape of cell: rod

Gram staining: negative

Motility: negative

Spore formation: negative

II. Physiological Property

TABLE 1 Items tested Property Reduction of nitrate NegativeDenitrification Negative Formation of indole Positive Formation ofhydrogen sulfide Weakly positive (zinc acetate test paper method)Hydrolysis of starch Positive Utilization of citrate: Simmons's citratemedium Negative Christensen's citrate Positive medium Formation ofpigment Forms insoluble yellow to orange pigment Urease Negative OxidasePositive Catalase Positive Growth at 37° C. Positive Growth at 42° C.Negative Behavior for oxygen Aerobic growth but not anaerobic O-F testOxidative formation of acid from glucose Hydrolysis of casein PositiveHydrolysis of gelatin Positive Hydrolysis of DNA Positive Hydrolysis ofesculin Positive Growth in McConkey's medium Negative VP reactionNegative Acid formation from saccharides: L-Arabinose Positive (no gasformation) D-Xylose Weakly positive (no gas formation) D-GlucosePositive (no gas formation) Maltose Positive (no gas formation) SucrosePositive (no gas formation) Lactose Negative Trehalose Positive (no gasformation) D-Mannitol Positive (no gas formation) Inositol NegativeGlycerol Weakly positive (no gas formation) Soluble starch Positive (nogas formation)

In this connection, this enzyme can be distinguished from knowntransglutaminase, because it does not have the activity to catalyzeformation of isopeptide between glutamine residue and lysine residue inprotein, namely transglutaminase activity. It can also be distinguishedfrom known protease, because it does not have the activity to hydrolyzepeptide bond of protein, namely protease activity.

Regarding the culturing method of the above strain for the production ofthe protein-deamidating enzyme, either a liquid culturing or a solidculturing, but preferably a liquid culturing, may be used. The liquidculturing can be carried out for example in the following manner.

Any medium can be used with the proviso that a microorganism capable ofproducing the protein-deamidating enzyme can grow in the medium.Examples of the medium to be used include those which contain carbonsources such as glucose, sucrose, glycerol, dextrin, molasses andorganic acids, nitrogen sources such as ammonium sulfate, ammoniumcarbonate, ammonium phosphate, ammonium acetate, peptone, yeast extract,corn steep liquor, casein hydrolysate and beef extract and inorganicsalts such as potassium salts, magnesium salts, sodium salts, phosphoricacid salts, manganese salts, iron salts and zinc salts.

The medium pH is adjusted to a value of approximately from 3 to 9,preferably from about 5.0 to 8.0, and the culturing is carried out underaerobic conditions at a temperature of generally from about 10 to 50°C., preferably from about 20 to 37° C., for a period of from 12 hours to20 days, preferably from 1 to 7 days. As the culturing method, a shakingculture method or an aerobic submerged jar fermentor culture method maybe used.

The protein-deamidating enzyme of the invention can be obtained byisolating the protein-deamidating enzyme from the thus obtained culturebroth in the usual way. For example, when the protein-deamidating enzymeis isolated and purified from the culture broth, purifiedprotein-deamidating enzyme can be obtained by treating it in the usualway by the combination of centrifugation, UF concentration, salting outand various types of chromatography such as of an ion exchange resin.

The invention is described more illustratively. That is, theaforementioned Chryseobacterium sp. No. 9670 was used as aprotein-deamidating enzyme producing strain and cultured in a liquidmedium, and production, purification and properties of the enzyme wereexamined.

One loopful of cells grown on a fresh slant medium were inoculated intoLB Base medium (mfd. by Gibco) and cultured on a shaker at 25° C. for 2to 7 days, and then centrifugation supernatant is obtained.

After completion of the culturing, the enzyme of interest was purifiedby subjecting the culture broth to centrifugation (12,000 rpm, 4° C., 20minutes) to obtain the supernatant as a crude enzyme solution, andtreating the thus obtained solution by UF concentration (SEP-0013),salting out, Phenyl-Sepharose and Sephacryl S-100. Steps of thepurification are shown in Table 2.

TABLE 2 Total Specific protein Total activity Purification mg activity UU/mg Yield % degree Culture 3547.8 606.8 0.171 100 1.00 filtrate UF492.8 483.6 0.981 79.7 5.74 Concentrate Salting out 404.3 383.5 0.94963.2 5.55 Phenyl- 35.83 255.5 7.13 42.1 41.7 Sepharose Sephacryl 7.02236.4 33.7 39.0 197.1 S-100

In this case, measurement of the enzyme activity was carried out in thefollowing manner using Z-Gln-Gly and casein as substrates.

Activity measuring method: A 10 μl portion of each enzyme solution isadded to 100 μl of 176 mM phosphate buffer (pH 6.5) containing 10 mMZ-Gln-Gly and incubated at 37° C. for 60 minutes, and then the reactionis stopped by adding 100 μl of 12% trichloroacetic acid solution. Aftercentrifugation (15,000 rpm, 4° C., 5 minutes), the resulting supernatantis measured in the following manner using F-kit ammonia (mfd. byBoehringer-Mannheim) (A1). Separately, the same measurement is carriedout using water instead of the enzyme solution (A2).

A 10 μl portion of the supernatant and 190 μl of water are added to 100μl of the F-kit ammonia reagent 2, the resulting mixture is allowed tostand at room temperature for 5 minutes and then the absorbance of 100μl portion of the reaction solution is measured at 340 nm (E1). Theremaining 200 μl portion is mixed with 1.0 μl of reagent 3 (glutamatedehydrogenase), allowed to stand at room temperature for 20 minutes andthen the absorbance of the remaining 200 μl is measured at 340 nm (E2).

The amount of enzyme which releases 1 μmol of ammonia within one minuteunder the above reaction conditions is defined as one unit andcalculated based on the following formula.U/ml=1.76×[A1(E1−E2)−A2(E1−E2)]

Using 1% casein (Hamerstein, mfd. by Merck) instead of 10 mM Z-Gln-Glyas the substrate, the activity is measured in the same manner to confirmthat the enzyme acts upon amino groups bonded to the protein. In thiscase, the protease activity was also checked by measuring the absorbanceof the centrifugation supernatant after termination of the reaction at280 nm. The amount of enzyme which increases 1 OD units under thiscondition was defined as one unit of protease activity.

Transglutaminase activity was measured by the following hydroxamic acidmethod using Z-Gln-Gly as the substrate.

-   -   Reagent A 0.2 M Tris-HCl buffer (pH 6.0)        -   0.1 M hydroxylamine        -   0.01 M reduced-type glutathione        -   0.03 M benzyloxycarbonyl-L-glutaminylglycine    -   Reagent B 3 N hydrochloric acid        -   12% trichloroacetic acid        -   5% FeCl₃.6H₂O (dissolved in 0.1 N HCl)

A 1:1:1 mixture of these solutions is used as reagent B.

A 0.05 ml portion of each enzyme solution is mixed with 0.5 ml of thereagent A to carry out 10 minutes of the reaction at 37° C., thereaction solution is mixed with 0.5 ml of the reagent B to stop thereaction and to effect formation of Fe complex, and then the absorbanceat 525 nm is measured. As a control, the same reaction is carried outusing the same enzyme solution heat-inactivated in advance, and theabsorbance is measured to calculate its difference from the absorbanceof the intact enzyme solution. Separately, a calibration curve isprepared using L-glutamic acid γ-monohydroxamate instead of the enzymesolution, for use in the calculation of the amount of formed hydroxamicacid based on the just described difference in absorbance, and theenzyme activity which forms 1 μmol of hydroxamic acid within one minuteis defined as one unit.

In this connection, the amount of protein was determined using BCAProtein Assay Kit (mfd. by Pierce) and bovine serum albumin as thestandard protein.

(1) Measurement of molecular weight: This was 20 kDa when measured bySDS-polyacrylamide gel electrophoresis (FIG. 1).

(2) Measurement of optimum pH: A 100 μl portion of 40 mMBritton-Robinson buffer solution (having a pH value of from 3 to 12)containing 10 mM Z-Gln-Gly was pre-incubated at 37° C. for 5 minutes, 10μl of each enzyme solution containing 0.32 μg of the protein-deamidatingenzyme was added to the buffer and incubated at 37° C. for 60 minutes tomeasure the enzyme activity. As the results, the optimum pH was around6.

(3) Measurement of optimum temperature: A 10 μl portion of enzymesolution containing 1.21 μg of the protein-deamidating enzyme was addedto 100 μl of a substrate solution [176 mM phosphate buffer (pH 6.5)containing 10 mM Z-Gln-Gly], and the reaction was carried out at eachtemperature for 60 minutes to measure the enzyme activity. As theresults, the optimum temperature was around 60° C.

(4) Measurement of pH stability: A 22 μl portion of enzyme solutioncontaining 0.75 μg of the protein-deamidating enzyme (in 40 mMBritton-Robinson buffer solution having a pH value of from 3 to 12) wastreated at 30° C. for 18 hours. Thereafter, the remaining enzymeactivity was measured. As the results, the enzyme was stable atapproximately from pH 5 to 9.

(5) Measurement of temperature stability: A 43 μl portion of enzymesolution containing 1.76 μg of the protein-deamidating enzyme [in 50 mMphosphate buffer solution (pH 7.0)] was allowed to stand at eachtemperature for 10 minutes, and then the remaining enzyme activity wasmeasured. As the results, the enzyme was stable at up to 50° C.

(6) Substrate specificity: Each solution of various proteins having afinal concentration of 1% (50 mM phosphate buffer, pH 6.5) was used asthe substrates and mixed with the protein-deamidating enzyme, and 1 hourof the reaction was carried out at 37° C. After the reaction,trichloroacetic acid solution was added to a final concentration of 6.4%to terminate the reaction, and the reaction mixture was centrifuged at13,000 rpm for 3 minutes to measure amount of ammonia in the resultingsupernatant. As a control, the same treatment was carried out by addingthe enzyme after termination of the reaction, and amount of ammonia inthe supernatant was measured. By subtracting the amount of releasedammonia by the control test from the amount of ammonia released by theenzyme reaction test, the amount of ammonia released by the enzymereaction was obtained to calculate ammonia releasing rate. The ammoniareleasing rate was expressed as the amount of ammonia released by 1 mgof enzyme during 1 minute. The results are shown in Table 3. When aportion of the reaction mixture after completion of the reaction wassubjected to SDS-PAGE and compared with the control, increased ordecreased molecular weight of the protein was not found. This resultmeans that the enzyme of the invention is a novel enzyme which can bedistinguished from known transglutaminase and protease.

TABLE 3 Ammonia releasing rate Protein (μmole/min/mg ± SD^(a)) α-Casein,bovine milk 19.12 ± 0.51  β-Casein, bovine milk 18.11 ± 0.15 α-Lactoalbumin, bovine milk 0.836 ± 0.009 β-Lactoglobulin, bovine milk0.728 ± 0.001 Bovine serum albumin, bovine 0.009 ± 0.001 Ovalbumin,chichen egg 0.005 ± 0.002 Gluten, wheat^(b) 7.200 ± 0.333 Gliadin,what^(b) 5.473 ± 0.017 Zein, corn^(b) 0.655 ± 0.176 Soy protein isolate1.170 ± 0.064 Collagen, Type I, bovine 0.177 ± 0.017 Achilles tendon^(b)Gelatin, Type B, bovine skin 0.696 ± 0.100 Muscle acetone powder, 0.210± 0.034 chicken breast^(b) Myoglobin, horse skeletal 0.014 ± 0.001muscle Actin, bovine muscle 0.450 ± 0.022 RNase A, bovine pancreas 2.912± 0.367 α-Chymotrypsinogen A, bovine 0.650 ± 0.118 pancreas Aprotinin,bovine lung 0.224 ± 0.064

(7) Measurement of isoelectric point: When measured by theelectrofocusing Ampholine (600 V, 4° C., 48 hours), isoelectric point ofthis enzyme was 10.0.

Next, the method of the invention for the modification of protein usingthe protein-deamidating enzyme is described in detail.

The protein-deamidating enzyme of the invention is allowed to act uponvarious proteins. Any type of protein can be used, with the proviso thatit undergoes action of the enzyme, and its examples are plant proteinsobtained from beans and cereals and animal proteins which include milkproteins such as casein and β-lactoglobulin, egg proteins such asovalbumin, meat proteins such as myosin and actin, blood proteins suchas serum albumin and tendon proteins such as gelatin and collagen. Alsoincluded are partially hydrolyzed proteins obtained by chemicaltreatment with an acid or an alkali or by enzymatic treatment forexample with a protease, chemically modified proteins with variousreagents and synthesized peptides.

These substrate proteins are subjected to the reaction in the form ofsolution, slurry or paste, but their concentrations are not particularlylimited and optionally selected depending on the desired properties andconditions of the deamidating protein of interest. Also, the solution,slurry or paste of each substrate protein is not limited to an aqueoussolution and may be in the form of emulsion with oil and fat and, asoccasion demands, may contain additives such as salts, saccharides,proteins, perfumes, moisture keeping agents and coloring agents.

The reaction conditions such as amount of the enzyme, reaction time andtemperature and pH of the reaction solution are not particularly limitedtoo, but the reaction may be generally carried out using the enzyme inan amount of from 0.1 to 100 units, preferably from 1 to 10 units, basedon 1 g of protein, at a reaction temperature of from 5 to 80° C.,preferably from 20 to 60° C., at a reaction solution pH of from 2 to 10,preferably from 4 to 8, and for a period of from 10 seconds to 48 hours,preferably from 10 minutes to 24 hours. In addition, these conditionscan be optionally changed depending, for example, on the purity of theenzyme to be used and the kind and purity of the substrate protein to beused.

Thus, the action of the protein-deamidating enzyme of the invention uponvarious proteins renders possible direct deamidation of amido groups inthe protein. As the results, negative charge of the thus deamidatedprotein increases which accompanies reduced pI, increased hydrationability and increased electrostatic repulsion. Also, changes in thehigher-order structure of protein result in the increased surfacehydrophobic property. These effects result in the improvement offunctionality of protein, such as increased solubility anddispersibility, increased foamability and foam stability and increasedemulsification ability and emulsification stability.

Thus, the protein having improved functionality greatly expands its usemainly in the field of food. A number of plant proteins show poorfunctionality such as solubility, dispersibility and emulsificationability particularly under weakly acidic condition which is the pH rangeof general food, so that they are limited in the use in many foodarticles which include acidic drinks such as coffee whitener and juice,dressing, mayonnaise and cream. However, when a plant protein havingpoor solubility, such as wheat gluten for example, is deamidated by theinvention, its solubility and dispersibility are increased, so that itsuse in these unsuited food articles becomes possible and it can be usedas tempura powder having high dispersibility.

The enzyme of the invention can also be used for the improvement ofdough in the field of bakery and confectionery. For example, a doughhaving high gluten content has problems in terms of handling andmechanical characteristics of the dough because of its lowextensibility, as well as volume and quality of the finished bread.These problems can be resolved by improving the extensibility throughthe deamidation of gluten with this enzyme. In addition, since thedeamidated gluten shows the effect as an emulsifying agent, breadproducing characteristics such as keeping quality and softness are alsoimproved. Also, a dough containing deamidated gluten has low plasticityand excellent extensibility, so that this is suitable for the productionof crackers, biscuits, cookies, pizza pies or crusts of pie, and theenzyme of the invention can be used in their production. For thispurpose, the enzyme of the invention is used in an amount of from 0.01to 10,000 units, preferably from 0.1 to 150 units, based on the totalweight of dough comprised of wheat flour, water and the like materialswhich may be mixed in the usual way.

Still more, when a protein in food, which causes allergy, anon-resistant disease or a genetic disease, is treated with the enzymeof the invention, its toxicity and allergenic property can be removed orreduced. In the case of food allergy, most of the allergen peptidesgenerally have high hydrophobic property. When they are converted intohydrophilic peptides by their treatment with the enzyme, the allergenicproperty is removed or reduced. Particularly, large effect can beobtained when an allergen peptide contains glutamine residue such as thecase of a wheat gluten allergen.

Still more, when a protein is deamidated by this enzyme,mineral-sensitivity of the protein is reduced, so that the solublemineral content in a protein-mineral solution is increased andabsorption of minerals in the human body can be improved. It is wellknown in general that absorption of calcium contained in food by thehuman body is improved when calcium is solubilized using an organic acidor casein phosphopeptide. Based on the same mechanism, it is possible tosolubilize a large quantity of calcium by deamidation of the protein bythe enzyme of the invention. Using the deamidated protein, high mineral(e.g., calcium)-containing drinks and mineral (e.g., calcium) absorptionenhancing agents can be produced.

Also, in the case of the production of amino acid based condiments(hydrolysate of animal protein (HAP) and hydrolysate of plant protein(HVP)), bean paste (miso) or soy sauce, other effects such as reductionof bitterness, improvement of protein hydrolyzing ratio by protease andincrease in the glutamic acid content can be obtained. It is well knownin general that the cause of bitterness is originated from hydrophobicpeptides, so that the deamidation renders possible reduction of bitterpeptides. It is known also that a peptide having glutamic acid on itsN-terminal has the effect to mask bitterness. In addition, since primarystructure and higher-order structure of a material protein are changedby deamidation, protease-sensitivity of the protein can also beincreased. As the results, the low protein hydrolyzing ratio, as aproblem involved in the enzymatic production of HAP and HVP, can also beimproved. On the other hand, reduction of the glutamic acid contentcaused by the formation of pyroglutamic acid is another problem in theproduction of HAP and HVP. Pyroglutamic acid is formed by theintramolecular cyclization of free glutamine, but it can be prevented bydeamidation of the material protein and, as the result, the glutamicacid content is increased.

Still more, the enzyme of the invention can be used as an agent for usein the control of the transglutaminase reaction. Transglutaminase isbroadly used as a protein modifying agent, namely as a cross-linkingenzyme, in the field of food and other industrial fields. The purpose ofthe use of transglutaminase is to obtain gelled protein products by theprotein cross-linking reaction of the enzyme or to improve functionalityof protein, but it is difficult to obtain a product having desiredcross-linking degree and functionality in response to respective use andobject, namely to control the cross-linking reaction such as terminationof the reaction at an appropriate stage. Particularly in the case of themodification of proteins for food use, it is not desirable to addgenerally known transglutaminase inhibitors such as EDTA, ammoniumchloride and SH reagents.

It is possible to terminate the transglutaminase reaction by adding theprotein-deamidating enzyme of the invention at a desired stage duringthe reaction of transglutaminase. That is, the transglutaminase reactioncan be stopped by converting glutamine residues which are the target ofthe transglutaminase reaction in the substrate protein into glutamicacid residues by the protein-deamidating enzyme.

In that case, it is necessary that the affinity of theprotein-deamidating enzyme for glutamine residues in a protein as itssubstrate is higher than that of transglutaminase, but the latter caseof reaction requires the ε-amino group of lysine in addition toglutamine residues while the former case requires only water other thanthe glutamine residues, which is abundantly present in the reactionenvironment, so that it can be assumed that the reaction ofprotein-deamidating enzyme generally precedes the reaction oftransglutaminase. As a matter of course, a modified or gelled proteinhaving desired cross-linking degree can be obtained by appropriatelytreating a substrate protein with the protein-deamidating enzyme toeffect conversion of desired glutamine groups into glutamic acidresidues and then subjecting the thus treated protein to thetransglutaminase reaction.

It can also be used as a reagent for use in the functional modificationof protein, namely for use in protein engineering. When the substrateprotein is an enzyme, enzyme-chemical and physicochemical properties ofthe enzyme can be modified. For example, when an enzyme protein isdeamidated by the enzyme of the invention, isoelectric point of theenzyme protein is reduced so that its pH stability can be modified.Also, other properties of the enzyme such as substrate affinity,substrate specificity, reaction rate, pH-dependency,temperature-dependency and temperature stability can be modified bychanging the structure or electric environment of its active site.

It also can be used as reagents for analyses and studies of protein,such as a reagent for use in the determination of amide content ofprotein and a reagent for use in the solubilization of protein.

In addition, it can be used for the improvement of extraction andconcentration efficiencies of cereal and bean proteins. In general,proteins of cereals and beans such as wheat and soybean are mostlyinsoluble in water and cannot therefore be extracted easily, but suchproteins can be extracted easily and high content protein isolates canbe obtained when these proteins are solubilized by treating a suspensionof wheat flour or soybean flour with the enzyme of the invention.

In the case of soybean protein, when the protein is generally extractedfrom defatted soybean powder or flakes (protein content, about 50%), theprotein is firstly insolubilized by a heat treatment, an ethanoltreatment or an isoelectric point treatment at around pH 4.5, and thensoluble polysaccharides are removed to obtain a soybean proteinconcentrate having a protein content of about 70%. When protein of morehigher purity is desired, it is prepared by suspending or dissolvingsoybean powder or the concentrate in a dilute alkali solution todissolve the protein and then removing insoluble substances. Thisproduct is called soybean protein isolate and contains about 90% of theprotein. These soybean protein products are applied to various foodarticles such as ham, sausages and baby food, utilizing functions ofsoybean protein, such as emulsifying activity, gelling property andwater-retaining property as well as its high nutritive value.

When the enzyme of the invention is used in producing these soybeanprotein products, not only the yield is improved due to the increasedsolubility of protein but also high concentration protein products canbe produced. Since the protein products obtained in this manner aredeamidated, they have excellent functionality. In consequence, they canexert excellent effects when used in various food articles such as meator fish products and noodles, and their use renders possible productionof food articles having new texture and functionality.

The following describes the protein-deamidating enzyme of the invention,a gene which encodes the protein-deamidating enzyme, a recombinantvector which contains the gene, a transformant transformed with thevector and a method for the production of the protein-deamidatingenzyme, which comprises culturing the transformant in a medium, therebyallowing the transformant to produce the protein-deamidating enzyme, andsubsequently collecting the protein-deamidating enzyme from the culturemixture.

Regarding the protein-deamidating enzyme of the invention, all of theprotein-deamidating enzymes which can be obtained by theprotein-deamidating enzyme production processes are included (i.e.,allelic mutants and allelic variants are included), in whichparticularly preferred one is a polypeptide which has the amino acidsequence of SEQUENCE NO. 6 shown in the Sequence Listing attached,wherein one or more of amino acid residues of the amino acid sequencemay be modified by at least one of deletion, addition, insertion andsubstitution, and more preferred one is a polypeptide which has theamino acid sequence of SEQUENCE NO. 6 shown in the Sequence Listing.

Examples of the gene which encodes the protein-deamidating enzyme of thepresent invention include a gene which can be obtained from amicroorganism capable of producing the protein-deamidating enzyme bycloning of the gene and a gene which has a certain degree of homologywith the gene. Regarding the homology, a gene having a homology of atleast 60% or more, preferably a gene having a homology of 80% or moreand more preferably a gene having a homology of 95% or more can beexemplified. The following nucleotide (polynucleotide; DNA or RNA) isdesirable as the gene which encodes the protein-deamidating enzyme ofthe invention.

A nucleotide which comprises a nucleotide selected from the followingnucleotides (a) to (g) and which encodes a polypeptide having theactivity to deamidate amido groups in protein;

-   (a) a nucleotide which encode a polypeptide having the amino acid    sequence of SEQUENCE NO. 6 shown in the Sequence Listing,-   (b) a nucleotide which encode a polypeptide having the amino acid    sequence of SEQUENCE NO. 6 shown in the Sequence Listing, wherein    one or more of amino acid residues of the amino acid sequence are    modified by at least one of deletion, addition, insertion and    substitution,-   (c) a nucleotide which has the nucleotide sequence of SEQUENCE NO. 5    shown in the Sequence Listing,-   (d) a nucleotide which has the nucleotide sequence of SEQUENCE NO. 5    shown in the Sequence Listing, wherein one or more of bases of the    nucleotide sequence are modified by at least one of deletion,    addition, insertion and substitution,-   (e) a nucleotide which hybridizes with any one of the above    nucleotides (a) to (d) under a stringent condition,-   (f) a nucleotide which has homology with any one of the above    nucleotides (a) to (d), and-   (g) a nucleotide which is degenerate with respect to any one of the    above nucleotides (a) to (f).

The gene which encodes the protein-deamidating enzyme of the inventioncan be prepared from the microorganism capable of producing theprotein-deamidating enzyme by carrying out cloning of the gene in thefollowing manner. Firstly, the protein-deamidating enzyme of theinvention is isolated and purified from a microorganism capable ofproducing the protein-deamidating enzyme by the aforementioned methodand information on its partial amino acid sequence is obtained.

Regarding the determination method of a partial amino acid sequence, itis effective to carry out a method in which purified protein-deamidatingenzyme is directly applied to an amino acid sequence analyzer (such asProtein Sequenser 476A, manufactured by Applied Biosystems) by Edmandegradation method [J. Biol. Chem., vol. 256, pp. 7990-7997 (1981)], ora method in which its limited hydrolysis is carried out using aproteolytic enzyme, the thus obtained peptide fragments are isolated andpurified and then amino acid sequences of the thus purified peptidefragments are analyzed.

Based on the information of the thus obtained partial amino acidsequences, the protein-deamidating enzyme gene is cloned. In general,the cloning is carried out making use of a PCR method or a hybridizationmethod.

When a hybridization method is used, the method described in [MolecularCloning, A Laboratory Manual, edit. by T. Maniatis et al., Cold SpringHarbor Laboratory, 1989] may be used.

When a PCR method is used, the following method can be used.

Firstly, a gene fragment of interest is obtained by carrying out PCRreaction using genomic DNA of a microorganism capable of producing theprotein-deamidating enzyme as the template and synthetic oligonucleotideprimers designed based on the information of partial amino acidsequences. The PCR method is carried out in accordance with the methoddescribed in [PCR Technology, edit. by Erlich H. A., Stockton Press,1989]. When nucleotide sequences of the thus amplified DNA fragments aredetermined by a usually used method such as the dideoxy chaintermination method, a sequence which corresponds to the partial aminoacid sequence of the protein-deamidating enzyme is found in the thusdetermined sequences, in addition to the sequences of syntheticoligonucleotide primers, so that a part of the protein-deamidatingenzyme gene of interest can be obtained. As a matter of course, a genewhich encodes complete protein-deamidating enzyme can be cloned byfurther carrying out a cloning method such as the hybridization methodusing the thus obtained gene fragment as a probe.

In Example 11 of this specification, a gene coding for theprotein-deamidating enzyme was determined by the PCR method usingChryseobacterium sp. No. 9670. Complete nucleotide sequence of the genecoding for the protein-deamidating enzyme originated fromChryseobacterium sp. No. 9670 is shown in the SEQUENCE NO. 5, and theamino acid sequence encoded thereby was determined to be the sequenceshown in the SEQUENCE NO. 6. In this connection, there are countlessnucleotide sequences which correspond to the amino acid sequence shownin the SEQUENCE NO. 6, in addition to the nucleotide sequence shown inthe SEQUENCE NO. 5, and all of these sequences are included in the scopeof the invention.

The gene of interest can also be obtained by chemical synthesis based onthe information of the amino acid sequence shown in the SEQUENCE NO. 6and the nucleotide sequence shown in the SEQUENCE NO. 5 (cf., Gene,60(1), 115-127 (1987)).

Regarding the protein-deamidating enzyme gene of the invention, anucleotide which encodes a polypeptide having the amino acid sequenceshown in SEQUENCE NO. 6, wherein one or more of amino acid residues ofthe amino acid sequence are modified by at least one of deletion,addition, insertion and substitution, a gene which hybridizes with thenucleotide under a stringent condition, a nucleotide which has homologywith the nucleotide and a nucleotide which is degenerate with respectiveto the nucleotide are also included in the invention, with the provisothat the polypeptides encoded thereby have the protein-deamidatingenzyme activity.

The term “under stringent condition” as used herein means the followingcondition. That is, a condition in which the reaction system isincubated at a temperature of from 50 to 65° C. for a period of from 4hour to overnight in 6×SSC (1×SSC is a solution composed of 0.15 M NaCland 0.015 M sodium citrate, pH 7.0) containing 0.5% SDS, 5× Denhart's [asolution composed of 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone and 0.1% Ficoll 400] and 100 μg/ml of salmon sperm DNA.

By using the entire portion or a part of the protein-deamidating enzymegene whose complete nucleotide sequence has been revealed making use ofChryseobacterium sp. No. 9670, as a probe for hybridization, DNAfragments having high homology with the protein-deamidating enzyme geneof SEQUENCE NO. 5 can be selected from genomic DNA libraries or cDNAlibraries of microorganisms capable of producing otherprotein-deamidating enzymes.

The hybridization can be carried out under the aforementioned stringentcondition. For example, a genomic DNA library or a cDNA library obtainedfrom a microorganism capable of producing a protein-deamidating enzymeis fixed on a nylon membrane, and the thus prepared nylon membrane issubjected to blocking at 65° C. in a pre-hybridization solutioncontaining 6×SSC, 0.5% SDS, 5× Denhart's and 100 μg/ml of salmon spermDNA. Thereafter, each probe labeled with ³²P is added to the nylonmembrane which is then incubated overnight at 65° C. The thus treatednylon membrane is washed in 6×SSC at room temperature for 10 minutes, in2×SSC containing 0.1% SDS at room temperature for 10 minutes and then in0.2×SSC containing 0.1% SDS at 45° C. for 30 minutes, subsequentlysubjecting the thus washed membrane to an auto-radiography to detect aDNA fragment which specifically hybridizes with the probe. Also, geneswhich show various degree of homology can be obtained by changingcertain conditions such as washing.

On the other hand, primers for use in the PCR reaction can be designedfrom the nucleotide sequence of the gene of the invention. By carryingout the PCR reaction using these primers, gene fragments having highhomology with the gene of the invention can be detected and the completegene can also be obtained.

In order to determine whether the thus obtained gene encodes apolypeptide having the protein-deamidating enzyme activity of interest,the thus determined nucleotide sequence is compared with the nucleotidesequence coding for the protein-deamidating enzyme of the invention orwith its amino acid sequence, and the identity is estimated based on thegene structure and homology. Alternatively, it is possible to determinewhether the gene encodes a polypeptide which has the protein-deamidatingenzyme activity of interest by producing a polypeptide of the gene andmeasuring its protein-deamidating enzyme activity.

The following method is convenient for producing a polypeptide havingthe protein-deamidating enzyme activity using the protein-deamidatingenzyme gene of the invention.

Firstly, transformation of a host is carried out using a vectorcontaining the protein-deamidating enzyme gene of interest and thenculturing of the thus obtained transformant is carried out undergenerally used conditions, thereby allowing the strain to produce apolypeptide having the protein-deamidating enzyme activity.

Examples of the host to be used include microorganisms, animal cells andplant cells. Examples of the microorganisms include bacteria such asEscherichia coli and other species belonging to the genera Bacillus,Streptomyces and Lactococcus, yeast species such as of the generaSaccharomyces, Pichia and Kluyveromyces and filamentous fungi such as ofthe genera Aspergillus, Penicillium and Trichoderma. Examples of theanimal cells include those which utilize the baculovirus expressionsystem.

Confirmation of the expression and expressed product can be made easilyby the use of an antibody specific for the protein-deamidating enzyme,and the expression can also be confirmed by measuring theprotein-deamidating enzyme activity.

As described in the foregoing, purification of the protein-deamidatingenzyme from the transformant culture medium can be carried out byoptional combination of centrifugation, UF concentration, salting outand various types of chromatography such as of ion exchange resins.

In addition, since the primary structure and gene structure of theprotein-deamidating enzyme have been revealed by the invention, it ispossible to obtain a gene coding for the amino acid sequence of anatural protein-deamidating enzyme, in which one or more of amino acidresidues of the amino acid sequence are modified by at least one ofdeletion, addition, insertion and substitution, by introducing randommutation or site-specific mutation using the gene of the invention. Thismethod renders possible preparation of a gene coding for aprotein-deamidating enzyme which has the protein-deamidating enzymeactivity but its properties such as optimum temperature, temperaturestability, optimum pH, pH stability and substrate specificity areslightly changed, and it also renders possible production of suchprotein-deamidating enzymes by means of gene engineering techniques.

Examples of the method for introducing random mutation include achemical DNA treating method in which a transition mutation is inducedto convert cytosine base into uracil base by the action of sodiumhydrogensulfite [Proceedings of the National Academy of Sciences of theUSA, vol. 79, pp. 1408-1412 (1982)], a biochemical method in which basesubstitution is induced during the step of double strand formation inthe presence of [α-S] dNTP [Gene, vol. 64, pp. 313-319 (1988)] and a PCRmethod in which PCR is carried out by adding manganese to the reactionsystem to decrease accuracy of the nucleotide incorporation [AnalyticalBiochemistry, vol. 224, pp. 347-353 (1995)].

Examples of the method for introducing site-specific mutation include amethod in which amber mutation is employed [gapped duplex method;Nucleic Acids Research, vol. 12, no. 24, pp. 9441-9456 (1984)], a methodin which recognition sites of restriction enzymes are used [AnalyticalBiochemistry, vol. 200, pp. 81-88 (1992); Gene, vol. 102, pp. 67-70(1991)], a method in which mutation of dut (dUTPase) and ung (uracil DNAglycosylase) is used [Kunkel method; Proceedings of the National Academyof Sciences of the USA, vol. 82, pp. 488-492 (1985)], a method in whichamber mutation is induced using DNA polymerase and DNA ligase[oligonucleotide-directed dual amber: ODA) method: Gene, vol. 152, pp.271-275 (1995); JP-A-7-289262 (the term “JP-A” as used herein means an“unexamined published Japanese patent application”)], a method in whicha host introduced with a DNA repair system is used (JP-A-8-70874), amethod in which a protein capable of catalyzing DNA chain exchangereaction is used (JP-A-8-140685), a method in which PCR is carried outusing two different primers for mutation use to which recognition sitesof restriction enzymes are added (U.S. Pat. No. 5,512,463), a method inwhich PCR is carried out using a double-stranded DNA vector having aninactivated drug resistance gene and two different primers [Gene, vol.103, pp. 73-77 (1991)] and a method in which PCR is carried out makinguse of amber mutation (WO 98/02535).

Also, site-specific mutation can be introduced easily by the use ofcommercially available kits. Examples of such kits include Mutan™-G(manufactured by Takara Shuzo) in which the gapped duplex method isused, Mutan™-K (manufactured by Takara Shuzo) in which the Kunkel methodis used, Mutan™-Express Km (manufactured by Takara Shuzo) in which theODA method is used and QuickChange™ Site-Directed Mutagenesis Kit(manufactured by STRATAGENE) in which primers for mutation use andPyrococcus furiosus DNA polymerase are used, as well as TaKaRa LA PCR invitro Mutagenesis Kit (manufactured by Takara Shuzo) and Mutan™-SuperExpress Km (manufactured by Takara Shuzo) as kits in which PCR is used.

Thus, the primary structure and gene structure of theprotein-deamidating enzyme provided by the invention render possibleproduction of an inexpensive and high purity polypeptide having theprotein-deamidating enzyme activity by means of gene engineeringtechniques.

In this connection, various literature and references are cited in thespecification, and all of them are incorporated herein by references.

Examples of the present invention are given below by way of illustrationand not by way of limitation. Unless otherwise noted, the term “%” usedin the following means “W/V %”.

EXAMPLE 1 Screening of Protein-Deamidating Enzyme Producing Strain

a) Enrichment culturing: Each of 320 soil samples was inoculated into 5ml of medium A containing Cbz-Gln-Gly as the sole nitrogen source andcultured at 30° C. for 6 days on a shaker. A 50 μl portion of theculture medium was inoculated in fresh medium A and again cultured at30° C. for 3 days on a shaker. The culture broth was spread or streakedon a nutrient medium, and the grown bacterium or fungus was isolated asa single colony.

b) Plate selection: The thus obtained colonies were replicated on anagar medium comprised of the medium A and agar and cultured at 30° C.for 6 days, and the thus grown strains (150 bacterial strains and 294fungal strains) were picked up.

c) Check of protein-deamidating enzyme productivity: Each of thesestrains was inoculated into a lactose liquid medium and cultured at 30°C. for 2 to 7 days on a shaker, and the culture broth was centrifuged toobtain culture supernatant. When protein-deamidating enzyme activity inthese culture supernatant was measured, 50 bacterial strains and 85fungal strains were positive.

-   -   Medium A: 0.1% Cbz-Gln-Gly, 0.5% glucose, 0.02% KH₂PO₄, 0.02%        MgSO₄.7H₂O, 0.01% NaCl, 0.002% CaCl₂, 0.0002% FeSO₄.7H₂O,        0.0005% NaMo₄.2H₂O, 0.0005% NaWO₄.4H₂O, 0.0005% MnSO₄.4H₂O and        0.01% CuSO₄.5H₂O (pH 8.0).    -   Lactose liquid medium: 0.5% lactose, 1.0% peptone, 0.17%        Na₂HPO₄.H₂O, 0.025% KH₂PO₄, 0.025% MgSO₄.7H₂O and 0.005%        FeSO₄.7H₂O (pH 7.2, adjusted with 6 N NaOH).

EXAMPLE 2

From the 50 bacterial strains and 85 fungal strains obtained in Example1, one strain (No. 9670) was selected to carry out the following test.As described in the specification, this was identified as a strainbelonging to the genus Chryseobacterium.

Chryseobacterium sp. No. 9670 was cultured on a shaker at 25° C. for 64hours using the aforementioned LB Base medium. Time course of theculturing is shown in Table 4.

TABLE 4 Culture time (h) pH Growth Enzyme activity (U/ml) 18 8.24 ++0.116 40 8.65 +++ 0.224 64 9.41 +++ 0.201

EXAMPLE 3

From the 50 bacterial strains and 85 fungal strains obtained in Example1, one strain (No. 9671) was selected to carry out the following test.This was also identified as a strain belonging to the genusChryseobacterium. Chryseobacterium sp. No. 9671 was cultured in the samemanner as described in Example 2. Protein-deamidating activity in theculture medium is shown in Table 5.

EXAMPLE 4

Each of Chryseobacterium indologenes IFO 14944, Chryseobacteriummeningosepticum IFO 12535, Chryseobacterium balustinum IFO 15053,Flavobacterium aquatile IFO 15052, Empedobacter brevis IFO 14943,Sphingobacterium spiritivorum IFO 14948, Sphingobacterium heparinum IFO12017, Aureobacterium esteraromatidum IFO and Myroides odoratus IFO14945 was cultured in the same manner as described in Example 2.Protein-deamidating enzyme activity in each culture medium is shown inTable 5.

TABLE 5 Deamidating activity (U/ml) Culture Z-Gln- Strain time (h) GlyCasein Chryseobacterium sp. No. 9671 24 0.160 0.143 Chryseobacteriumgleum JCM 2410 20 0.130 0.108 Chryseobacterium indologenes IFO 14944 200.070 0.019 Chryseobacterium meningosepticum 31 0.067 0.017 IFO 12535Chryseobacterium balustinum IFO 15053 16 0.111 0.025 Flavobacteriumaquatile IFO 15052 48 0.019 0.038 Empedobacter brevis IFO 14943 20 0.0400.149 Sphingobacterium spiritivorum IFO 14948 20 0.057 0.078Sphingobacterium heparinum IFO 12017 48 0.047 0.031 Aureobacteriumesteraromatidum IFO 3751 31 0.003 0.019 Myroides odoratus IFO 14945 410.005 0.026

Production of protein-deamidating enzyme was confirmed in each strain.

EXAMPLE 5

When cultured in the same manner using M 17 medium (mfd. by Difco),Tryptone Soya medium (mfd. by Oxioid) or heart infusion medium (mfd. byDifco) instead of LB medium, production of the protein-deamidatingenzyme was confirmed by all of the strains used in Examples 2 to 4.

EXAMPLE 6

The culture broth obtained after 40 hours of culturing in Example 2 wassubjected to 20 minutes of centrifugation at 4° C. and at 12,000 rpm(22,200×g) to remove cells, and the thus obtained centrifugationsupernatant was concentrated to about 25 times using an ultrafiltrationmembrane (SEP-0013, mfd. by Asahi Chemical Industry) and thenfreeze-dried to obtain a crude enzyme powder. This was dissolved in 10mM sodium phosphate buffer solution (pH 6.5) containing 2.0 M NaCl, thethus formed insoluble matter was removed by 15 minutes of centrifugationat 4° C. and at 10,000 rpm (12,300×g), and the thus obtainedcentrifugation supernatant was applied to a Phenyl-Sepharose CL-6Bcolumn (mfd. by Pharmacia) which had been equilibrated with 10 mM sodiumphosphate buffer solution (pH 6.5) containing 2.0 M NaCl, and theadsorbed protein was eluted by a linear NaCl density gradient of from2.0 M to 0 M.

Fractions having protein-deamidating activity were combined,concentrated using the ultrafiltration membrane and then applied to aSephacryl S-100 column which had been equilibrated with 10 mM sodiumphosphate buffer solution (pH 6.5) containing 0.6 M NaCl and 0.05% Tween20, and the elution was carried out using the same buffer. Fractionshaving protein-deamidating activity were combined and concentrated usingthe ultrafiltration membrane to obtain a protein-deamidating enzymesolution. Results of the purification are shown in Table 2.

When the purified preparation of protein-deamidating enzyme obtained inthis manner was subjected to 4 to 12% SDS-polyacrylamide gelelectrophoresis, this was confirmed to be a single protein having amolecular weight of 20 kDa as shown in the lane 2 of FIG. 1.

When measured by the aforementioned assay methods (the method which usesZ-Gln-Gly as the substrate and the method which uses casein as thesubstrate), the thus obtained enzyme preparation showed the activitiesof 33.7 units/ml (Z-Gln-Gly as the substrate) and 13.5 units/ml (caseinas the substrate). Transglutaminase activity and protease activity werenot detected.

EXAMPLE 7 Preparation of Deamidated Proteins

A 1 g portion of wheat gluten, milk caseinate, whey protein, albumenprotein or soybean protein was suspended in 100 ml of 100 mM sodiumphosphate buffer (pH 6.5), 6.13 units of the protein-deamidating enzymewas added to the suspension and the mixture was allowed to undergo theenzyme reaction at 37° C. for 24 hours on a shaker. Time course of thechanges in deamidation ratio during this reaction is shown in FIG. 2.The deamidation ratio was expressed as percentage of ammonia or ammoniumreleased in the solution, determined after completion of the reaction,based on the total amido content in the protein. The total amido contentin protein was obtained by hydrolyzing the protein (2% w/v) at 110° C.for 3 hours in 3 N sulfuric acid and determining amount of the releasedammonia. It can be understood that the deamidation reaction wasgenerated in the enzyme-added reaction system, because ammonia wasreleased with the lapse of the reaction time, while release of ammoniawas not observed in the reaction carried out in the absence of theenzyme as a control. The deamidation ratio was 74% in wheat gluten, 60%in caseinate, 23% in whey protein, 20% in soybean protein and 7% inalbumen protein. After the reaction, this was heated at 75° C. for 15minutes to terminate the reaction by deactivating the enzyme, dialyzedagainst water and then freeze-dried to obtain deamidated protein powder.Also, the reaction product obtained by carrying out in the absence ofthe enzyme as a control was treated in the same manner to obtain acontrol powder.

FIG. 3 shows patterns of the 4 to 12% SDS-polyacrylamide gelelectrophoresis of these deamidated proteins and control proteins. Itcan be understood that molecular weights of the deamidated proteins(lanes 3, 6, 9 and 12) were not changed in comparison with theenzyme-untreated control proteins, namely that both degradation of theproteins and increase in their molecular weight by cross-linking werenot generated. In this case, although a slight shift of protein band wasobserved in the deamidated caseinate (lane 3) or soybean protein (lane12) to higher molecular weight side, it is considered that this shiftwas due to the increase in minus charge in protein by deamidation, whichcaused reduction of its binding to SDS, also having minus charge, byelectrostatic repulsion, and thereby resulted in the reduction of totalminus charge of the protein-SDS complex in comparison with thenon-deamidated protein, thus entailing reduced migration in theelectrophoresis.

EXAMPLE 8 Improvement of Functionality (Foam Characteristics) ofDeamidated Protein

Each of the deamidated protein powders obtained in Example 7 andenzyme-untreated protein powders obtained by the control test wasdissolved in 10 mM phosphate buffer (pH 7.0) to a concentration of 0.5mg/ml, and foamability and foam stability were measured bymicro-conductivity method (Wilde P J, Colloid and Interface Science,178, 733-739, 1996). The foam stability was expressed as the remainingdegree of conductivity after 5 minutes. The results are shown in Table6.

TABLE 6 Foam Foamability stability Protein (%) (%) Control gluten —*8.05 Deamidated gluten 1.25 41.44 Control albumen protein 1.50 33.96Deamidated albumen protein 1.89 36.79 Control soybean protein isolate1.84 42.96 Deamidated soybean protein isolate 2.81 63.19 *Not measurabledue to considerably poor foamability.

Thus, it is evident that foam characteristics of protein can be sharplyimproved by deamidating the protein with the enzyme of the invention.

EXAMPLE 9 Improvement of Functionality (Emulsifying Characteristics) ofDeamidated Protein

A 4 ml portion of solution of each of the deamidated protein powdersobtained in Example 7 and enzyme-untreated protein powders obtained bythe control test (1.0 mg/ml in 10 mM phosphate buffer (pH 7.0) and 1.0 gof corn oil (manufactured by Sigma) were put into a vial, pre-agitatedfor 1 minute using Vortex mixer and then passed five times through asingle pass bulb homogenizer (EmulsiFlex-20000-B3, Avesten, Ottawa,Canada) under a high pressure (200 kPa), thereby preparing an oil/wateremulsion. Particle distribution of the fresh emulsion was measured usinga laser diffraction particle size analyzer (Coulter LS230, Coulter,Hialesh, FL). The emulsifying activity was expressed by specific surfacearea (surface area of particles per 1 ml of emulsion). Regarding theemulsifying stability, degrees of creaming, flocculation and coalescencewhich indicate disintegration of emulsion were observed with the nakedeye after 7 days of standing at room temperature. Results of theemulsifying activity and emulsifying stability are shown in Tables 7 and8, respectively.

TABLE 7 Emulsifying activity Protein (cm²/ml) Control gluten 13,120Deamidated gluten 57,531 Control casein 41,040 Deamidated casein 67,068Control whey protein 29,534 Deamidated whey protein 29,996 Controlalbumen protein 37,252 Deamidated albumen protein 58,238 Control soybeanprotein isolate 16,512 Deamidated soybean protein isolate 30,796

TABLE 8 Protein Creaming Flocculation Coalescence Control gluten − −++++++ Deamidated gluten − − + Control casein ++ − ± Deamidated casein +− ± Control whey protein +++ − − Deamidated whey protein +++ − − Controlalbumen protein ++++ ++++ − Deamidated albumen protein ++ ± − Controlsoybean protein isolate ++++ + + Deamidated soybean protein ++ + ±isolate

Thus, it is evident that emulsifying activity and emulsifying stabilityof protein can be sharply improved by deamidating the protein with theenzyme of the invention.

EXAMPLE 10 Improvement of Functionality (Solubility) of DeamidatedProtein

Each of the deamidated protein powders obtained in Example 7 andenzyme-untreated protein powders obtained by the control test wassuspended and dissolved in 10 mM citrate-phosphate buffer having a pHvalue of from 2.7 to 6, 10 mM Tris-HCl buffer having a pH value of from7 to 9 or an aqueous solution of pH 12 (adjusted with NaOH) to aconcentration of 2.0 mg/ml, shaken at room temperature for 30 minutesand then allowed to stand at room temperature for 30 minutes. Aftermeasuring the pH value, this was centrifuged at a high speed of16,000×g, the thus obtained supernatant was filtered through a 0.45 μmmembrane and then the protein content in the filtrate was measured bythe BCA method. Using the protein content in the filtrate as an index ofsolubility, the solubility at pH 12 was shown as 100% (Methods ofTesting Protein Functionality, pp. 47-55, edited by G. M. Hall, BlackieAcademic & Professional, London, 1996). The pH-solubility curves ofdeamidated proteins are shown in FIGS. 4 to 8.

Thus, it is evident that solubility of protein can be sharply improvedby deamidating the protein with the enzyme of the invention.

In the case of wheat gluten, the enzyme-untreated protein showed almostno solubility from pH 4.5 to about pH 9.0, while the deamidated wheatgluten showed about 40% solubility at pH 4.8 and 80% or more solubilityat pH 6 or higher, which means remarkable improvements in the solubility(FIG. 4). In the case of caseinate, the enzyme-untreated caseinateshowed low solubility at the weakly acidic range (pH 5 to pH 6) which isthe pH of usual foods (e.g., 22% at pH 5.4). On the other hand, thedeamidated caseinate showed about 30% solubility at pH 5.1 and about 70%or more solubility at pH 5.3 or higher (FIG. 5). In the case of wheyprotein, the enzyme-untreated protein showed no solubility from pH 3.8to about pH 4.7, while the deamidated whey protein showed about 50%solubility at pH 3.9 and at pH 4.7 (FIG. 6). In the case of soybeanprotein, the solubility of the enzyme-untreated protein is generally lowand showed only about 10% at pH 6.6 and about 30% at pH 7.5. On theother hand, the deamidated soybean protein showed 70% or more solubilityat pH 6 or higher, which means remarkable improvements in the solubility(FIG. 7). In the case of egg protein, the enzyme-untreated proteinshowed no solubility from pH 3.9 to pH 4.3, while the deamidated proteinshowed about 70% solubility at pH 3.9 and about 43% solubility at pH 4.3(FIG. 8).

EXAMPLE 11 Isolation of Gene Coding for the Protein-Deamidating EnzymeDerived from Chryseobacterium sp. No. 9670

In this specification, gene manipulation techniques were carried out inaccordance with textbooks (e.g., “Molecular Cloning” 2nd ed., ColdSpring Harbor Laboratory Press, 1989) unless otherwise noted.

a) Isolation of Chromosomal DNA

A 4.5 ml portion of chromosomal DNA solution having a concentration of210 μg/ml was obtained from 100 ml of culture broth in accordance withthe method described in “Current Protocols in Molecular Biology”, Unit2.4 (John Wiley & Sons, Inc., 1994).

b) Determination of Partial Amino Acid Sequence

The purified protein-deamidating enzyme obtained in Example 3 wasapplied to a protein sequenser (mfd. by Applied Biosystems) to determinean N-terminal amino acid sequence of 20 residues shown in SEQUENCENO. 1. Next, the purified protein-deamidating enzyme obtained in Example3 was reduced and alkylated using performic acid and then hydrolyzedwith trypsin. The thus obtained hydrolysate was applied to a reversephase liquid chromatography, and one of the separated peptide fractionswas applied to the protein sequenser to determine an internal amino acidsequence of 20 residues shown in SEQUENCE NO. 2.

SEQUENCE NO. 1: Leu-Ala-Ser-Val-Ile-Pro-Asp-Val-Ala-Thr-Leu-Asn-Ser-Leu-Phe-Asn-Gln-Ile-Lys-Asn SEQUENCE NO. 2:Ser-Pro-Ser-Asn-Ser-Tyr-Leu-Tyr-Asp-Asn-Asn-Leu-Ile-Asn-Thr-Asn-Cys-Val-Leu-Thrc) Preparation of DNA Probe by PCR

Based on the N-terminal region amino acid sequence and the internalamino acid sequence, the following two mixed oligonucleotides weresynthesized using a DNA synthesizer (mfd. by Applied Biosystems) andused as PCR primers.

SEQUENCE NO. 3 Sense primer:5′-GCI(TA)(CG)IGTIAT(TCA)CC(TACG)GA(TC)GT-3′ <N-1g> SEQUENCE NO. 4Antisense primer: 5′-A(AG)(AGTC)AC(AG)CA(AG)TT(AGTC)GT(AG)TT(AGT)AT-3′ <M-2a>

Using these primers and the Chryseobacterium sp. No. 9670 chromosomalDNA as the template, PCR reaction was carried out using Omnigene ThermalCycler (mfd. by Hybaid) under the following conditions.

<PCR reaction solution> 10 × PCR reaction buffer (mfd. by Perkin Elmer)5.0 μl dNTP mixture solution (each 2.5 mM, mfd. by Promega) 4.0 μl 20 μMsense primer 10.0 μl  20 μM antisense primer 10.0 μl  distilled water20.25 μl  chromosomal DNA solution (190 μg/ml) 0.5 μl Taq DNA polymerase(mfd. by Perkin Elmer) 0.25 μl 

<PCR reaction condition> Stage 1: denaturation (94° C., 5 minutes)  1cycle Stage 2: denaturation (94° C., 1 minute) 30 cyles annealing (44°C., 1 minute) elongation (72° C., 1 minute) Stage 3: elongation (72° C.,10 minutes)  1 cycle

When the thus obtained DNA fragment of about 0.48 kb was cloned intopCRII (mfd. by Invitrogene) and then its nucleotide sequence wasdetermined, a nucleotide sequence coding for the aforementioned partialamino acid sequence was found in a region just after the sense primerand just before the antisense primer. This DNA fragment was used as aDNA probe for use in the cloning of the complete gene.

d) Preparation of Gene Library

As a result of the Southern hybridization analysis of theChryseobacterium sp. No. 9670 chromosomal DNA, a single band of about4.9 kb capable of hybridizing with the probe DNA was found in an EcoRIdigest. In order to carry out cloning of this EcoRI DNA fragment ofabout 4.9 kb, a gene library was prepared in the following manner. Thechromosomal DNA prepared in the above step a) was digested with EcoRI,and the thus obtained digest was ligated to an EcoRI-treated λ ZAPII(mfd. by Stratagene) vector and packaged using Gigapack III Gold (mfd.by Stratagene) to obtain the gene library.

e) Screening of Gene Library

The 0.48 kb DNA fragment obtained in the above step c) was labeled usingMegaprime DNA Labeling System (mfd. by Amersham) and ³²P-α-dCTP. Usingthis as a probe, the gene library obtained in the above step d) wasscreened by plaque hybridization. Phage particles were recovered fromthe thus obtained positive plaques, and then a plasmid p9T1-2 containingan EcoRI fragment of about 4.5 kb was obtained by the in vivo excisionmethod in accordance with the instruction provided by Stratagene.

f) Determination of Nucleotide Sequence

Nucleotide sequence of the plasmid p9T1-2 was determined in theconventional way. The nucleotide sequence which encodes theprotein-deamidating enzyme is shown in SEQUENCE NO. 5. Also, amino acidsequence encoded by the SEQUENCE NO. 5 is shown in SEQUENCE NO. 6. TheN-terminal region amino acid sequence (SEQUENCE NO. 1) and internalamino acid sequence (SEQUENCE NO. 2) determined in the above step b)were found in this amino acid sequence.

SEQUENCE NO. 5 TTTAAAACGATCCTGACAAAAGAAGTAAAAGGGCAAACCAATAAATTGGCGAGTGTAATTCCTGATGTAGCTACATTAAATTCTTTATTCAATCAAATAAAGAATCAGTCTTGCGGTACCTCTACGGCGTCCTCACCATGCATCACATTCAGATATCCTGTAGACGGATGTTATGCAAGAGCCCATAAGATGAGACAAATCTTAATGAACAACGGCTATGACTGTGAAAAACAATTTGTATACGGAAACCTAAAGGCATCAACAGGAACTTGCTGTGTGGCGTGGAGCTACCACGTTGCAATATTGGTAAGCTATAAAAATGCTTCCGGAGTAACGGAAAAAAGAATTATTGATCCTTCACTATTTTCAAGCGGTCCTGTAACAGATACAGCATGGAGAAACGCTTGCGTTAACACCTCTTGCGGATCTGCATCCGTTTCCTCTTATGCTAATACTGCAGGAAATGTTTATTACAGAAGTCCTAGTAATTCTTACCTGTATGACAACAATCTGATCAATACCAACTGTGTACTGACTAAATTTTCACTGCTTTCCGGATGTTCTCCTTCACCTGCACCGGATGTATCCAGCTGTGGATTT (555 bp) SEQUENCE NO.6 L A S V I P D V A T L N S L F N Q I K N Q S C G T S T A S S P C I T FR Y P V D G C Y A R A H K M R Q I L M N N G Y D C E K Q F V Y G N L K AS T G T C C V A W S Y H V A I L V S Y K N A S G V T E K R I I D P S L FS S G P V T D T A W R N A C V N T S C G S A S V S S Y A N T A G N V Y YR S P S N S Y L Y D N N L I N T N C V L T K F S L L S G C S P S P A P DV S S C G F (185 amino acid)

The open reading frame of this gene is shown in SEQUENCE NO. 7. As shownin SEQUENCE NO. 7, the entire portion is encoded as a prepro proteinhaving 320 amino acid residues, in which N-terminal 135 residues(underlined in SEQUENCE NO. 7) correspond to the prepro region and theremaining 185 residues correspond to the mature protein (cf. SEQUENCENO. 6).

Among the 135 residues of the prepro region, the N-terminal 21 residueshave the characteristics of the signal sequence and therefore areconsidered to be the pre region, and the remaining 114 residues areconsidered to be the pro region.

The invention is not particularly limited to polypeptides havingprotein-deamidating activity and nucleotides encoding the same, but alsoincludes the longer polypeptides comprising the polypeptides havingprotein-deamidating activity (such as prepro proteins and pro proteins)and nucleotides encoding the same.

SEQUENCE NO. 7 AGTTAAAATAACCAACCAACTTAACAAAAACTCACCATTAAACTACAAATTACAATTATTATGAAAAATCTTTTTTTATCAATGATGGCCTTTGTGACCG          M  K  N  L  F  L  S  M  M  A  F  V  T  VTCTTAACTTTTAATTCCTGTGCCGATTCCAACGGGAATCAGGAAATCAAC  L  T  F  N  S  C  A  D  S  N  G  N  Q  E  I  N  GGAAAGGAAAAACTAAGTGTAAATGATTCTAAGCTGAAAGATTTCGGAAAG  K  E  K  L  S  V  N  D  S  K  L  K  D  F  G  K GACTGTACCGGTAGGGATAGACGAAGAAAACGGAATGATAAAGGTGTCAT T  V  P  V  G  I  D  E  E  N  G  M  I  K  V  S  FTTATGTTAACTGCGCAATTCTATGAAATTAAGCCGACCAAAGAAAATGAG  M  L  T  A  Q  F  Y  E  I  K  P  T  K  E  N  E  CAGTATATCGGAATGCTTAGACAGGCTGTTAAGAATGAATCTCCTGTACAQ  Y  I  G  M  L  R  Q  A  V  K  N  E  S  P  V  H CATTTTCTTAAAGCCTAATAGCAATGAAATAGGAAAAGTGGAGTCTGCAA I  F  L  K  P  N  S  N  E  I  G  K  V  E  S  A  SGTCCGGAAGACGTAAGATATTTTAAAACGATCCTGACAAAAGAAGTAAAA  P  E  D  V  R  Y  F  K  T  I  L  T  K  E  V  K  GGGCAAACCAATAAATTGGCGAGTGTAATTCCTGATGTAGCTACATTAAAG  Q  T  N  K  L  A  S  V  I  P  D  V  A  T  L  NTTCTTTATTCAATCAAATAAAGAATCAGTCTTGCGGTACCTCTACGGCGT S  L  F  N  Q  I  K  N  Q  S  C  G  T  S  T  A  SCCTCACCATGCATCACATTCAGATATCCTGTAGACGGATGTTATGCAAGA  S  P  C  I  T  F  R  Y  P  V  D  G  C  Y  A  RGCCCATAAGATGAGACAAATCTTAATGAACAACGGCTATGACTGTGAAAAA  H  K  M  R  Q  I  L  M  N  N  G  Y  D  C  E  KACAATTTGTATACGGAAACCTAAAGGCATCAACAGGAACTTGCTGTGTGG Q  F  V  Y  G  N  L  K  A  S  T  G  T  C  C  V  ACGTGGAGCTACCACGTTGCAATATTGGTAAGCTATAAAAATGCTTCCGGA  W  S  Y  H  V  A  I  L  V  S  Y  K  N  A  S  GGTAACGGAAAAAAGAATTATTGATCCTTCACTATTTTCAAGCGGTCCTGTV  T  E  K  R  I  I  D  P  S  L  F  S  S  G  P  VAACAGATACAGCATGGAGAAACGCTTGCGTTAACACCTCTTGCGGATCTG T  D  T  A  W  R  N  A  C  V  N  T  S  C  G  S  ACATCCGTTTCCTCTTATGCTAATACTGCAGGAAATGTTTATTACAGAAGT  S  V  S  S  Y  A  N  T  A  G  N  V  Y  Y  R  SCCTAGTAATTCTTACCTGTATGACAACAATCTGATCAATACCAACTGTGTP  S  N  S  Y  L  Y  D  N  N  L  I  N  T  N  C  VACTGACTAAATTTTCACTGCTTTCCGGATGTTCTCCTTCACCTGCACCGG L  T  K  F  S  L  L  S  G  C  S  P  S  P  A  P  DATGTATCCAGCTGTGGATTTTAATTAATTGATAATTTTACAGCACCTGCT  V  S  S  C  G  F               320 CATTTACAGAATCAGCAGGTGCTGTTATAT(1080) * SEQUENCE NO. 8 M K N L F L S M M A F V T V L T F N S C A D S NG N Q E I N G K E K L S V N D S K L K D F G K T V P V G I D E E N G M IK V S F M L T A Q F Y E I K P T K E N E Q Y I G M L R Q A V K N E S P VH I F L K P N S N E I G K V E S A S P E D V R Y F K T I L T K E V K G QT N K L A S V I P D V A T L N S L F N Q I K N Q S C G T S T A S S P C IT F R Y P V D G C Y A R A H K M R Q I L M N N G Y D C E K Q F V Y G N LK A S T G T C C V A W S Y H V A I L V S Y K N A S G V T E K R I I D P SL F S S G P V T D T A W R N A C V N T S C G S A S V S S Y A N T A G N VY Y R S P S N S Y L Y D N N L I N T N C V L T K F S L L S G C S P S P AP D V S S C G F (320 amino acid)

EXAMPLE 12 Construction of Plasmid for Use in the Expression ofProtein-Deamidating Enzyme in E. coli

Based on the DNA sequences which encode the N-terminal region amino acidsequence and the C-terminal region amino acid sequence, the followingtwo oligonucleotides were synthesized using a DNA synthesizer (mfd. byApplied Biosystems) and used as PCR primers.

SEQUENCE NO. 9 Sense primer for mature protein expression use:5′-CCGAATTCTTGGCGAGTGTAATTCCTGATG-3′ SEQUENCE NO. 10 Sense primer forprepro protein expression use: 5′-CAGAATTCATGAAAAATCTTTTTTTATCAATGGCC-3′SEQUENCE NO. 11 Antisense primer: 5′-TCGAATTCTTAAAATCCACAGCTGGATAC-3′

Using these primers and the protein-deamidating enzyme gene-containingplasmid p9T1-2 as the template, PCR reaction was carried out usingOmnigene Thermal Cycler (mfd. by Hybaid) under the following conditions.

<PCR reaction solution> 10 × PCR reaction buffer (mfd. by Perkin Elmer)10.0 μl  dNTP mixture solution (each 2.5 mM, mfd. by Promega) 8.0 μl 20μM sense primer 2.5 μl 20 μM antisense primer 2.5 μl distilled water75.5 μl  plasmid p7T-1 solution (50 μg/ml) 1.0 μl Taq DNA polymerase(mfd. by Perkin Elmer) 0.5 μl

<PCR reaction condition> Stage 1: denaturation (94° C., 5 minutes) 1cycle Stage 2: denaturation (94° C., 1 minute) 30 cycles annealing (55°C., 1 minute) elongation (72° C., 1 minute) Stage 3: elongation (72° C.,10 minutes) 1 cycle

Each of the DNA fragment of about 0.57 kb obtained by the combination ofsense primer for mature protein expression use with antisense primer andthe DNA fragment of about 0.98 kb obtained by the combination of senseprimer for prepro protein expression use with antisense primer wascloned into pCRII (mfd. by Invitrogen) to confirm that the nucleotidesequence was correct, and then the DNA fragment of about 0.57 kb and theDNA fragment of about 0.98 kb were recovered from these plasmids byEcoRI treatment. Each of these DNA fragments was inserted into EcoRIsite of an expression vector pGEX-1λT for E. coli use (mfd. byPharmacia), and the protein-deamidating enzyme-encoding DNA wasconnected to the C-terminal-corresponding side of the glutathione Stransferase-encoding DNA contained in the pGEX-1λT, in the samedirection, thereby obtaining a plasmid pN7-9 containing the DNA fragmentcoding for the mature protein and a plasmid pP3-9 containing the DNAfragment coding for the prepro protein. These plasmids can express afusion protein of glutathione S transferase with protein-deamidatingenzyme under control of tac promoter, and the protein-deamidating enzymecan be cut off from the fusion protein by thrombin treatment.

EXAMPLE 13 Expression of Protein-Deamidating Enzyme in E. coli

A transformant was obtained by introducing each of the expressionplasmids pN7-9 and pP3-9 into E. coli BL21 (mfd. by Pharmacia). As acontrol, a transformant of E. coli BL21 having the expression vectorpGEX-1λT was also obtained. Each of these transformants was cultured at37° C. on a 200 rpm rotary shaker using LB medium containing 100 μg/mlof ampicillin, and the cells obtained at the logarithmic growth phase(OD₆₀₀=0.9−1.0) were mixed with 0.1 mM in final concentration of IPTG,cultured for 4 hours under the same conditions and then collected. Thethus collected cells were suspended in 1/10 volume culture broth of 50mM Tris-HCl (pH 8.0)/2 mM EDTA, mixed with 0.1 mg/ml in finalconcentration of egg white lysozyme and 0.1% in final concentration ofTriton X-100 and allowed to stand at 30° C. for 15 minutes, and then thethus formed viscous DNA was sheared by mild ultrasonic treatment (3cycles of 10 sec. on and 30 sec. off) to obtain a cell extract. A 100 μlportion of the cell extract was mixed with 4 μl of thrombin (1 U/μl in 9mM sodium phosphate (pH 6.5)/140 mM NaCl) and allowed to stand at roomtemperature for 16 hours to obtain thrombin-treated cell extract. Also,a sample obtained by adding 4 μl of a buffer solution (9 mM sodiumphosphate (pH 6.5)/140 mM NaCl) and carrying out the same reaction wasused as a control of the thrombin treatment.

The protein-deamidating enzyme activity of the thus obtained samples wasmeasured, with the results shown in Table 9.

TABLE 9 Protein-deamidating activity (mU/ml) Thrombin Substrate:Substrate: Sample Transformant treatment Z-Gln-Gly casein 1 E. coliBL21/pN7-9 − 31.10 16.99 2 E. coli BL21/pN7-9 + 37.32 20.67 3 E. coliBL21/pP3-9 − 1.05 2.75 4 E. coli BL21/pP3-9 + 1.54 3.40 5 E. coliBL21/pGEX-1λT − 0.00 0.00 6 E. coli BL21/pGEX-1λT + 0.00 0.00

Thus, it is apparent that the E. coli strain having the matureprotein-deamidating enzyme expression plasmid pN7-9 expresses theprotein-deamidating activity. The E. coli strain having the preproprotein-deamidating enzyme expression plasmid pP3-9 also expressed theprotein-deamidating activity though at a low level. On the contrary,expression of the protein-deamidating activity was not found in thecontrol E. coli strain having the expression vector pGEX-1λT.

Separately, each of the samples 1, 2, 5 and 6 was subjected to 12%SDS-polyacrylamide gel electrophoresis to carry out Western blottinganalysis using an antibody specific for the protein-deamidating enzyme.As a result, a band which reacted with the antibody was detected in thesample 1 at a position of about 43 Da in molecular weight which seemedto be a fusion protein of glutathione S transferase with the matureprotein-deamidating enzyme, and a band was detected in the sample 2 at aposition of about 20 kDa in molecular weight corresponding to the matureprotein-deamidating enzyme, in addition to the band of about 43 Da inmolecular weight. On the other hand, a band capable of reacting with theantibody was not detected in the samples 5 and 6.

On the basis of these results, it was confirmed that a recombinantprotein-deamidating enzyme can be produced in E. coli using theprotein-deamidating enzyme gene obtained by the invention.

A novel enzyme capable of acting upon amido groups in protein andthereby catalyzing the deamidation reaction, which was not found in theprior art, was found for the first time from a microorganism which isadvantageous for industrial production. A broad range of applicationsare expected by this enzyme.

Also, since the primary structure and gene structure of theprotein-deamidating enzyme were provided by the invention, inexpensiveand high purity production of polypeptide having protein-deamidatingenzyme activity by gene engineering techniques became possible.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese patent application No. Hei.11-345044 filed on Dec. 3, 1999, both herein incorporated by reference.

1. An isolated enzyme which has an activity to deamidate amido groups ina protein, wherein said enzyme comprises the amino acid sequence of SEQID NO:6.
 2. An isolated enzyme which has an activity to deamidate amidogroups in a protein by directly acting upon the amido groups withoutcutting peptide bonds and without cross-linking a protein, wherein saidenzyme comprises the amino acid sequence of SEQ ID NO:6.
 3. Arecombinant polypeptide having an action to deamidate amido groups inprotein, which is obtained by culturing a transformant transformed witha recombinant vector, which contains a nucleotide being selected fromthe following nucleotides (i) to (iii) and encoding a polypeptide havingan activity to deamidate amido groups in protein; (i) a nucleotide whichencodes a polypeptide having the amino acid sequence of SEQ ID NO:6,(ii) a nucleotide which has the nucleotide sequence of SEQ ID NO:5,(iii) a nucleotide which is degenerate with respect to any one of theaforementioned nucleotides (i) to (ii).
 4. A composition for use inmodification of a protein or a peptide, which comprises an isolatedenzyme having an activity to deamidate amido groups in protein orpeptide by directly acting upon the groups without causing severing ofpeptide bond and cross-linking of protein, as the active ingredient,wherein said enzyme comprises the amino acid sequence of SEQ ID NO:6. 5.An isolated enzyme which has an activity to deamidate amido groups inprotein, wherein said enzyme comprises the amino acid sequence encodedby the nucleotide sequence of SEQ ID NO:5.
 6. An isolated enzyme whichhas an activity to deamidate amido groups in protein by directly actingupon the groups without causing severing of peptide bond andcross-linking of protein, wherein said enzyme comprises the amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:5.
 7. Acomposition for use in modification of a protein or a peptide, whichcomprises an isolated enzyme having an activity to deamidate amidogroups in protein or peptide by directly acting upon the groups withoutcausing severing of peptide bond and cross-linking of protein, as theactive ingredient, wherein said enzyme comprises the amino acid sequenceencoded by the nucleotide sequence of SEQ ID NO:5.